Process for the preparation of β-keto aliphatic acid ester

ABSTRACT

The present invention provides a process for the preparation of antimicrobial compound β-keto aliphatic acid ester, a novel, aliphatic, fatty acid derivative from  Bacillus  sp. IICT-001.

FIELD OF THE INVENTION

The present invention relates to a process for preparation of β-keto aliphatic acid ester. The present invention particularly relates to a process for the preparation of antimicrobial compound β-keto aliphatic acid ester, a novel, aliphatic, fatty acid derivative from Bacillus sp. IICT-001.

BACKGROUND OF THE INVENTION

Fatty acid derivatives, which are mainly of fungal and bacterial metabolites, are subdivided into saturated and unsaturated type of compounds. A number of compounds have been isolated from Bacillus sp. in the art, such as iso-13 methyl tetradecanoic acid, fatty acid (Arch. Vetr. Ital 20,215 1969) peptide (Shoji et al., J. Antibiotics activity (Aszalos et al, J. Chromatography, 37, 477, 1968). The antibacterial pseudomonic acid produced by a Pseudomonas species represents an interesting new type of fatty acid ester (Berdy, CRC Hand Book of Antibiotic Compounds Vol VI p-391 1980). It contains several unusual chemical features in this family, such as epoxy, pyran and 9-hydroxy nonanic acid constituents. These compounds are normally soluble in nonpolar organic solvents such as hydrocarbons. Hydroxy acid was isolated primarily from bacterial species (Berdy, CRC Hand Book of Antibiotic Compound Vol II, 35, 1980). Antimicrobial and antitumor activity of natural fatty acids derived from different kinds of microbes including fungi, Actinomycetes or Myxobacteria, is a frequently reported property of such type of compounds. Fatty acid esters (Glycerides) such as monoolein, monolaurin, eoixenotid, pseudomonic acid A₁-B were isolated from different microbes with varied antimicrobial activity.

Cyclic acylpeptide, halobacillin from Bacillus species 11-cycloheptyl-2hydroxy undecanoic acid from thermo acidic Bacillus sp, sarcinic acid from different Bacillus sp. have been reported by earlier workers (Comprehensive natural products chemistry Vol 8 1986).

OBJECTS OF THE INVENTION

The main object of the present invention is to provide a process for the preparation of β-keto aliphatic avid ester, useful as therapeutic agent, a novel aliphatic acid ester from Bacillus species IICT-001 to isolate novel antimicrobial compounds from microbes, by screening many organisms from different soil samples and isolated a new strain designated as IICT-001 to produce potent antimicrobial compounds.

Another object of the present invention is the isolation, separation, purification and practical structure elucidation of a new bioactive, aliphatic, β-keto acid ester for the first time from a new strain of Bacillus sp . IICT 001.

Still another object of the present invention is that the new source for a new antimicrobial compound β-keto aliphatic acid ester, showing very good broad spectrum antimicrobial activity may be used as a new antibiotic with simple isolation process and might result in a new potent source of pharmaceutically active drug.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a process for preparation of β-keto aliphatic acid ester which comprises: growing a Bacillus sp. IICT 001 in growth medium for a period of at least 3-4 days to obtain broth, extracting the said broth with organic solvent, removing the solvent and purifying the β-keto aliphatic acid ester compound.

The new strain “Bacillus sp. IICT 01” was deposited at the Imtech Depository, in Chandigarh, India, which is a depository recognized under the Budapest Treaty. Applicants have deposited the strain using the same name and strain number used in the specification. The strain has also been identified by C.A.B. International Mycological Institute using Specimen No. 51 (May 9, 1988) Herb. IMI No. B12681.

In an embodiment of the invention the growth medium used is selected from the group consisting of nutrient medium and mineral salts medium.

In another embodiment of the invention the growth medium is supplemented with protein and carbon content such as soyabean meal, corn steep liquor, casein, casein hydrolysate glucose, malt extract.

In yet another embodiment of the invention, the growth of strain is carried out at a temperature range of 20 to 40° C. and a pH in the range of 4.5-7.5.

In another embodiment of the invention the solvents for extraction of broth used comprises a chlorinated organic solvent selected from the group consisting of chloroform, dichloromethane, dichloroethane, and ethyl acetate; or a polar solvent selected from the group consisting of methanol, ethanol and a mixture thereof.

In yet another embodiment of the invention the chromatographic method used comprises thin layer chromatography using silica gel as stationary phase and 1:1 methanol CHCl₃ as mobile phase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for the preparation of β-keto aliphatic acid ester, a novel aliphatic β-keto acid ester from Bacillus sp. IICT-001 a strain to produce potent antimicrobial compound, isolated in our pursuit to identify novel antimicrobial compound from terrestrial soil sample of Hyderabad. The new strain is identified based on microscopic, morphological, physiological and biochemical characteristics described in tables as Bacillus sp., which was grown in soyameal supplemented broth at optimum pH and temperature and extracting the said bacterial culture broth with non-polar solvents such as chloroform and ethyl acetate in combination with methanol, ethanol to extract through the antimicrobial compound in a known manner to get a crude fraction and purifying the antibiotic from the said crude fraction by conventional chromatography to recover antibiotic β-keto aliphatic acid ester.

The new strain IICT-001 is easy to grow and maintain, and multiplies profusely on nutrient broth. The production of antibiotic is good in a conventional soyabean meal supplemented production medium constituting soyabean meal 10 g, glucose 10 g, sodium chloride 5 g, calcium carbonate 1 g, water 1 lt. at optimum pH and optimum temperature.

The present strain Bacillus species is a gram positive, moving rods with bulging sporangia. The temperature range for the growth is 20-45° C. and the pH range is 4.5-8.0. The sodium chloride tolerance is recorded to be 4.0-7.0% Table 1 shows the colony characters while Table 2 shows the physiological characteristics such as temperature, pH, and sodium chloride tolerance. Tables 3 and 4 show the biochemical properties and utilisation of carbon source for the strain respectively.

TABLE 1 Margin Smooth Elevations − Pigments − Grams reactions + Shape Rods Size Small celled Endospore + Position of spore C Sporangia bulging + Motility +

TABLE 2 Physiological characteristics of strain Bacillus sp IICT 001 1 Temperature range for growth 20-45° C. 2 Optimum temperature for antibiotic protection 20-40° C. 3 PH range for the growth 4.5-9.0° C. 4 Optimum pH for antibiotic protection 6-9° C. 5 Sodium chloride tolerance 2.5-8.0° C.

TABLE 3 Biochemical characteristics of strain Bacillus sp 1 Citrate utilisation Negative 2 Caesin hydrolysis Positive 3 Starch hydrolysis Positive 4 Urea hydrolysis Negative 5 H₂S production Negative 6 Catalase Positive

TABLE 4 Utilisation of carbon sources and acid production by strain Bacillus sp. IICT 001 Arabinose Negative Cellobiose Positive Dextrose Positive Fructose Positive Galactose Negative Lactose Negative Maltose Positive Sucrose Positive

Based on the above properties the strain IICT-001 is identified as Bacillus sp. It differs from the type Bacillus sp. in the following properties. Optimum pH is 5.5-8.0, optimum temperature is 30-45° C., there is no growth conditions, The present strain Bacillus sp 001 differs from known Bacillus sp, in that it produces novel antibiotic under optimum temperature, pH and growth conditions, The growth conditions are requirement of carbon and nitrogen source along with macro and micronutrients for optimum yield of antimicrobial compound. (Buchanan and Gibbons, Bergy's Manual of Determinative Bacteriology, 8th ED., P.540, 1974; Sonenshein et al, Bacillus sp. American society for Microbiol, Washington D.C.P. 13,1993).

The bioassay guided isolation and purification has resulted in yellow viscous, UV positive compound soluble in organic solvents identified as β-keto aliphatic acid ester based on its physical and spectral properties. The spectral properties are as follows.

UV max (MeoH): 225 ¹H NMR CDCI₃(80 MH2): 0.88 t (CH ₃); 1.25 s, br (CH ₂)n; 2.16s(COCH ₂); 3.68 s(COOCH₃) IR. ^(V)max(CHCl³): cm⁻¹ 1730 (ester), 1670 (Carbonyl).

This organism is very stable and the compound production is continuous without loss of activity. The compound produced is also stable at around 80° C. also.

The present invention provides a process for preparation of β-keto aliphatic acid ester, by growing a novel strain Bacillus sp. in production medium for a period of at least 3-4 days in a conventional manner to obtain broth, extracting the said broth with organic solvent, removing the solvent and purifying the acid ester compound by conventional chromatographic methods,

The growth medium used may be such as nutrient medium and mineral salts medium. The medium may be supplemented with protein and carbon content such as soyabean meal, corn steep liquor, caesin, caesin hydrolysate glucose, malt extract. Growth of strain may be carried out at a temperature range of 20 C to 40 C and pH in the range of 4.5-7.5. The solvents for extraction of broth used may be chlorinated organic solvents such as chloroform, dichloromethane, dichloroethane, and ethyl acetate, polar solvents such as methanol, ethanol and mixture thereof.

The chromatographic method used may be such as thin layer chromatography using silica gel as stationary phase and 1:1 methanol:CHCl₃ as mobile phase. The active band is eluted with the same solvent.

The process for the preparation of β-keto aliphatic acid ester, a novel aliphatic β-keto acid ester from Bacillus sp. IICT-001 a strain to produce potent antimicrobial compound, isolated in our pursuit to identify novel antimicrobial compound from terrestrial soil sample of Hyderabad. The new strain is identified based on microscopic, morphological, physiological and biochemical characteristics described in tables as Bacillus sp., which was grown in soyameal supplemented broth at optimum pH and temperature and extracting the said bacterial culture broth with non-polar solvents such as chloroform and ethyl acetate in combination with methanol, ethanol to extract through the antimicrobial compound in a known manner to get a crude fraction and purifying the antibiotic from the said crude fraction by conventional chromatography to recover antibiotic β-keto aliphatic acid ester.

The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

EXAMPLE: 1

4 day old culture broth (2 lt) of Bacillus sp. is prepared with Soya supplemented medium constituting soya bean meal 20 g, glucose 20 g, sodium chloride 10 g, CaCo₃ 2 g, water 2 l at optimum pH and optimum temperature and sterilized at 15 pounds pressure for 20 mts. After it is cooled to room temperature a 10% inoculum grown for 24 hr. in the same culture medium is transferred aseptically to the above production medium and kept on shaker at 150 RPM for 4 days at 28+2° C. is extracted thoroughly with 1.5 l of ethyl acetate. The active organic crude extract (572) mg is used for further purification. The active compound purified by TLC using silica gel and 10% MeOH in CHCl₃ as mobile phase and the active UV positive band having an RF value of 0.6400 is eluted with the same solvent to get an yellow viscous antimicrobial compound (Yield 23.7 mg) of formula 1,

EXAMPLE: 2

5 day old culture broth (3 lt) of Bacillus sp grown in soya amended broth grown on orbital shaker at 37° C. at 170 RPM is extracted thoroughly with 20% MeOH in CHCl₃. The organic extracts pooled evaporated under vacuum at 40° C. to get 853 mg of crude fraction, which is purified by chromatography using a mixture of MeOH:CHCl₃ and eluted with the same solvent system to get pure, active yellow viscous antimicrobial compound (yield 35.12 mg) of formula 1.

EXAMPLE: 3

6 day old culture broth (4 lt) of Bacillus sp. grown in soya amended medium at 35° C. at 170 RPM is extracted thoroughly with 5% CHCl₃:MeOH. The organic extracts pooled evaporated under vacuum at 37° C. to get 1.12 gm of crude fraction, which is purified by chromatography using silica gel as stationary phase and 10% methanol in chloroform as mobile phase. The active band is eluted with the same solvent to get pure, antibacterial, yellow viscous compound (yield 46.89 mg) of formula 1.

EXAMPLE: 4

3 days old culture broth (1 lt) of Bacillus sp. grown in amended medium at 30° C. at 160 RPM is extracted thoroughly with chloroform in combination with methanol. The organic extracts pooled, evaporated under vacuum at 36° C. to get 284 mg of crude fraction, which is purified by chromatography using silica gel as stationary phase and 5% methanol in chloroform as mobile phase. The active band is eluted with the same solvent to get pure, antibacterial, yellow viscous compound (yield 11.71 mg) of formula 1.

EXAMPLE: 5

7 day old culture broth (2.5 lt) of Bacillus sp. grown in soya amended medium at 32° C. at 150 RPM is extracted thoroughly with dichloromethane in combination with methanol. The organic extracts pooled, evaporated under vacuum at 36° C. to get 709 mg of crude fraction, which is purified by chromatography using silica gel as stationary phase and 5% methanol in chloroform as mobile phase. The active band is eluted with same solvent to get pure, antibacterial yellow viscous compound (yield 16.98 mg) of formula 1.

Anti Microbial Assay:

All the media used are from Hi Media, Bombay, India. Bacterial strains were grown on nutrient agar and suspended in Muller Hinton broth and fungal strains in sabouroud broth. A conventional two fold serial dilution method is employed to determine minimum inhibitory concentration (Jones et al, 1984;In: Lenette E H, Ballows et al., Manual of clinical Microbiol., 972-977, Washington D.C., American Society for Microbiol). The compound dissolved in acetone at 2 mg/ml concentration is diluted in respective broths in the range of 100-1.56 μg/ml culture grown at 37° C. for 20 h were used as inoculum (approximately 10⁵⁻⁶ CFU/ml). Test cultures were incubated at 37° C. for 24 h. All the results (average of triplicates) were presented in μg/ml (Table 5). The lowest concentration of antimicrobial agent that results in the complete inhibition of microorganism represents the minimum inhibitory concentration (MIC (μg/ml).

The activity against the following organisms have been tested and the minimum inhibitory concentration against test microbes is presented in Table 5.

TABLE 5 Antimicrobial activity of β-keto aliphatic acid ester from Baccilus sp. IICT 001 No. microoragnism Concentration of compound μg/ml 1 Arthrobacter citrius <50 2 Bacillus cereus <6.25 3 B. licheniformis <25 4 B. polymixa <12.5 5 B. pumilus <12.5 6 B. subtilis <25 7 Clostridium sp. <25 8 Staphylococcus aureus <12.5 9 Streptococcus sp. <25 10 Escherchia coli <3.12 11 Klebsiella aerogenes <12.5 12 Pseudomonas aureoginosa <6.25 13 P. putida <12.5 14 Salmonella typhimurium <6.25 15 Sarcina lutea <12.5 16 Nocardia sp. <25 17 Candida albicans <12.5 18 Saccharomyces cerevisiae <50 The main advantages of the present invention are:

-   1. The maintenance of the organism is easy and growth is good in     variety of nutrient media. -   3. The antimicrobial compound is isolatable at varied temperatures     (20-40° C.). -   4. The antimicrobial compound is active on variety of microorganisms     such as gram positive and gram negative bacteria and fungi. -   5. The compound is active at low concentration on many organisms. -   6. The organism is very stable and activity is reproducible for many     generations tested for years. -   7. The culture Bacillus sp. IICT 001 can be stored in refrigerator     safely without any loss of activity. 

1. An antibiotic compound β-keto aliphatic acid ester isolated from Bacillus sp. IICT 001 and possessing the following spectral properties UV max (MeoH): 225 ¹H NMR CDCI₃(80 MH ₂): 0.88 t (CH ₃); 1.25 s, br (CH ₂)n; 2.16s (COCH ₂); 3.68 s(COOCH₃) IR. ^(V)max(CHCl³): cm⁻¹ 1730 (ester), 1670 (Carbonyl), wherein the antibiotic compound is made by a process comprising: preparing an at least 3 to 4 day old culture broth of Bacillus sp. with a medium comprising soya; extracting the resulting culture medium with a solvent at a temperature between 20° C. and 45° C. and a pH between 4.5 and 8.0; purifying the medium with a solvent by chromatography; and eluting with the same solvent to obtain the antimicrobial compound.
 2. A pharmaceutical composition comprising an effective amount of a β-keto aliphatic acid ester isolated from Bacillus sp. IICT 001 and possessing the following spectral properties UV max (MeoH): 225 ¹H NMR CDCI₃(80 MH ₂): 0.88 t (CH ₃); 1.25 s, br (CH ₂)n; 2.16s (COCH ₂); 3.68 s(COOCH₃) IR. ^(V)max(CHCI³): cm⁻¹ 1730 (ester), 1670 (Carbonyl) in admixture with a therapeutically acceptable carrier wherein the β-keto aliphatic acid ester is made by a process comprising: preparing an at least 3 to 4 day old culture broth of Bacillus sp. with a medium comprising soya; extracting the resulting culture medium with a solvent at a temperature between 20° C. and 45° C. and a pH between 4.5 and 8.0; purifying the medium with a solvent by chromatography; and eluting with the same solvent to obtain the β-keto aliphatic acid ester. 